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NEUROGENESIS IN DISEASES OF OXIDATIVE STRESS
by
Stefanie Brooke Marquez
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PATHOBIOLOGY)
August 2011
Copyright 2011 Stefanie Brooke Marquez

Transient hypoxia induces cell death in area CA1 of the rat hippocampus, but spares cells in CA2/3. Activation of stem cells in the subgranular zone of the dentate gyrus, and their subsequent proliferation and migration into damaged regions of the brain may serve in the adult to replace lost neurons. A critical question is if these new neurons form appropriate connections and restore function. In rat organotypic hippocampal cultures cell death, maturation and migration of neural stem cells (NSCs) and neuronal progenitor cells (NPCs) were confirmed after exposure to transient hypoxia (6 hours) and reoxygenation. NSCs, NPCs, immature neurons, and more mature neurons were immunohistochemically detected with antinestin, doublecortin, Tuj-1, and NeuN, respectively, and cell proliferation with antiPCNA immunostain, and BrdU uptake. Using electrophysiological techniques, in hypoxic cultures synaptic signals were absent in CA1 24h after hypoxia,but returned by 72h. These studies suggest hippocampal brain cultures are sensitive to hypoxia, and respond with restoration of physiologic function by new neurons. ❧ Transient hypoxia selectively induces cell death in pyramidal neurons in the CA1 region of the rat hippocampus, sparing neurons in CA2 and CA3. Neural stem cells (NSCs) in the subgranular zone of the dentate gyrus serves as a reservoir to replace the lost neurons. Evidence from initial studies in organotypic hippocampal slice cultures suggests that newborn neurons, derived from these NSCs, proliferate and migrate to the damaged area and may differentiate and integrate into the existing brain circuitry. In rat hippocampal slice cultures, after exposure to six hours of hypoxia followed by reoxygenation, cell death occurs in CA1, and there is maturation and migration of NSCs and NPCs. Electrophysiologic field recordings also confirmed absence of synaptic responses after 24 hours reoxygenation, and a restoration of synaptic response in CA1 at 72h after hypoxia. However, in this system, it is unclear if the synaptic activity recorded is the result of integration of newborn neurons into the CA1, or recovery of some remaining neurons originally present in CA1 that may have been temporarily incapacitated from the hypoxic insult, or alternatively, a result of contributions of both sets of neurons. As an initial step, we examined two sources of GFP-labeled NSCs. First, using a retroviral construct with the GFP gene located under the CMV promoter control, endogenous stem cell migration and differentiation was monitored. Second, by transplanting exogenous, GFP-labeled rat hippocampal stem cells into the slice cultures prior to inducing hypoxia, we show that in contrast to normoxic controls, many of these cells migrate within the slice to the affected CA1 region, and co-express markers of neuronal progenitors (DCX) and also more differentiated immature neurons (âIII tubulin). These results provide a basis for defining physiologic connections but yet do not exclude repair contributions of pre-existing neurons. ❧ Throughout life, neurogenesis occurs in the subgranular layer of the hippocampus and the olfactory bulb in the adult human central nervous system (Alvarez-Buylla et al 1998; Kempermann et al 2004; Zhao et al 2008; Verret et al 2007; Imayoshi et al 2009). Acute insults, such as hypoxia or ischemia, may stimulate proliferation of neural stem cells (NSCs) and differentiation of neural progenitor cells (NPCs) of limited potential (Kokaia et al 2003; Panschison 2009; Clarke et al 2009; Miles and Kernie 2008; Steiner et al 2006). Less is known about the response of these cell types to chronic stimuli present during specific neurodegenerative processes. However, neurogenesis is altered in the hippocampus of individuals with Alzheimer’s Disease (AD) and in rodent models of AD (Jin et al 2003c; Waldau et al 2008; Donovan et al 2006; Haughey et al 2002; Zhang et al 2007; Rodriguez et al 1998; Yu et al 2009; Chevallier et al 2005). We define neurogenesis in several areas of the brain affected in incipient AD (IAD) and AD: the frontal subcortical white matter and periventricular zone (PVZ), adjacent to the cerebral cortex. Nestin and doublecortin (DCX)-immunoreactive cells were found in the post-mortem brains of individuals diagnosed with either moderate or severe AD or IAD from minimally-cognitively impaired (MCI) and mildly-impaired brains. DCX-positive cells often appear as part of a pair, with either one or both of the doublet expressing DCX. In contrast, fewer DCX-positive cells were observed in those regions in age-matched controls, and primarily as single cells. The transcription factor Sox2, which promotes amplification of stem/progenitor cells during the cell cycle (Episkopou 2005; Wang et al 2007), was also upregulated in IAD and AD brains compared to controls. Primitive neuroectodermal cells expressing βIV tubulin were also found in the PVZ of age-matched controls, but to a greater extent in IAD and AD PVZ. We also noted increased numbers of cells expressing epidermal growth factor receptor (EGFR), a marker for transit amplifying cells, in the most anterior part of the PVZ and in frontal cortex of IAD and AD brain, but rarely in controls. These results suggest an expansion of the transit amplifying cell pool, possibly via both symmetric and asymmetric division of NPCs in the AD-affected brain, and migration of progenitors into the diseased area. A small fraction of the stem/progenitor cells differentiated into glia, while the others displayed markers of immature neurons.

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NEUROGENESIS IN DISEASES OF OXIDATIVE STRESS
by
Stefanie Brooke Marquez
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PATHOBIOLOGY)
August 2011
Copyright 2011 Stefanie Brooke Marquez